The treatment of chronic Achilles tendinopathy remains challenging with a relatively high percentage of non-responders. There are many proposed treatment options, of which an eccentric exercise program is currently the first treatment of choice. Despite the good results on pain scores after eccentric exercises, a 5-year follow-up study showed that only ~40% of the patients were completely pain free and 48% had received one or more alternative treatments [1
]. In accordance with this, there is still a need for treatments that would improve the benefits of a structured eccentric exercise program. Recent studies indicate that both cardiometabolic [2
], as well as nutritional factors [4
] can modulate local tendon healing. Although clinical studies are still scarce, a recent International Olympic Committee (IOC) consensus statement on dietary supplements in high-performance athletes [6
] proposes that dietary supplementation containing gelatin or hydrolysed collagen could potentially be useful for athletic populations as increased intake of collagen-derived peptides has been shown to modulate collagen synthesis [7
] and reduce tendon- [8
] and joint-related pain [9
]. Others have shown that glycine, as the most abundant component of collagen hydrolysates [11
], has disease-modifying properties in both animal [12
] and in vitro [14
] models of tendinopathy. There is also first evidence, that collagen peptides might improve functional ankle properties in chronic ankle instability [15
]. Therefore, we aimed to study the potential clinical benefits of a specific hydrolysed collagen supplement as add-on therapy to 6 months of an eccentric calf-strengthening and well-structured return-to-running program.
It has been well established that increased (micro) vascularity is associated with chronic (painful) tendon lesions [16
]. As a change in (micro)vascularity might be a hinge for improved tendon architecture, our aim was to check whether the oral intake of collagen peptides influence microvascular changes in tendinopathic areas of the Achilles tendon using real-time contrast-enhanced ultrasonography (CEUS) [17
]. In a recently published study [18
], it was shown that CEUS provides a more objective and reproducible assessment of tendon microvascularity in comparison with power Doppler ultrasound. Furthermore, CEUS was also shown to be moderately associated with increased Achilles tendon symptoms. We hypothesized that improvements in tendon pain and function throughout the course of the intervention would be reflected by a reduction in CEUS-based tendon microvascularity. A previous ultrasonographic tissue characterisation study has shown that structural integrity is decreased in both Achilles tendons in people with unilateral Achilles tendinopathy [19
]. As dietary supplementation of hydrolysed collagen appears to have systemic effects on collagen-dense tissue [20
], we aimed to study both tendons of each individual patient, independent of whether participants had uni- or bilateral symptoms.
The main finding of this small-scale but well-controlled pilot study is that oral supplementation of sCPs with high glycine content may accelerate the clinical benefits of a well-structured calf-strengthening and return-to-running program in patients with uni- or bilateral chronic Achilles tendinopathy symptoms. Although the study was not powered to detect differences in VISA-A scores or return-to-running sport activities between the 2 groups, it is interesting to note that, independent of randomization order, both clinical outcome variables appear to improve more during the sCPs supplementation period. Although the differences between the 2 groups at 3 months may appear small, the mean change in VISA-A score in group AB of 12.6 (9.7; 15.5) points is well above the minimum clinically important difference (MCID) of 6.5 for Achilles tendinopathy patients [31
]. In our placebo group BA, VISA-A increased only by 5.3 (2.3; 8.3) points after 3 months, which is below the MCID of 6.5. After crossing over from sCP to placebo and vice versa, group AB and BA showed an opposite response with, subsequently, a significant increase in VISA-A of respectively 5.9 (2.8; 9.0) and 17.7 (14.6; 20.7). These clinical findings extend on two earlier studies [32
], that reported respectively a pain modulating and in vitro anti-inflammatory effect of a nutraceutical containing collagen, mucopolysaccharides and vitamin C. Furthermore, in a rodent model of collagenase-induced Achilles tendinopathy, a 5% glycine-rich diet for 3 weeks improved hydroxyproline, glyosaminoglycans and non-collagenous protein content of the Achilles tendon [12
]. Glycine has also been shown to improve collagen matrix organisation strength and tenocyte remodelling, most likely by modulating both TNF-alpha, matrix metalloproteases and the availability of collagen precursors [14
]. As the sCP supplement in the present study contains 22% of glycine [11
], it could be postulated that the increased intake of 1.1 g of glycine per day may have contributed to observed clinical improvements in VISA-A scores during sCP supplementation. Due to nature of the co-intervention, a true washout period to bring participants back to their baseline condition is in Achilles tendinopathy patients not possible. From a pharmacokinetic perspective, the serum hydroxyproline content following the ingestion of hydrolysed collagen peptides returns to baseline within 12 h [25
]. Nevertheless, any potential carry-over effects have been accounted for in our LMM-analyses by looking at interaction of the order and treatment effect across time.
Given the small scale of the study and unequal distribution of men and women in our 2 study groups, there is a potential of selection bias. Although the adaptability of tendon to loading differs in men and women [34
], we are not aware of any intervention study that specifically showed clinically relevant gender difference in VISA-A scores following a calf-strengthening program. To evaluate homogeneity of the data at baseline between the 2 groups a discriminant analysis was carried out. The data revealed no statistically significant differences between both study groups. Unfortunately, the relatively low number of female participants does not allow for a gender-based inference. As women may be more prone to develop Achilles tendinopathy [35
], future random-controlled trials (RCTs) should aim to include an equal number of men and women in both study arms.
In order to maximize any potential synergistic effect of orally ingested sCPs with bi-daily calf-strengthening exercises (as per Alfredson’s protocol) on tendon collagen synthesis, our participants were instructed to ingest the sCPs 30 min before each exercise session. About 15–30 min after oral ingestion of collagen hydrolysates, free and peptide forms of serum hydroxyproline levels significantly increase and reach a maximum after 30–60 min and are almost back to baseline level after 7–12 h [25
]. Although this was not investigated as a separate outcome measure, the small time window between the bi-daily intake of a sachet with 2.5 g of sCPs and the calf-strengthening exercise may have contributed to the high compliance rate observed in our trial. A recent proof-of-concept study by Shaw et al. (2017) showed a dose-dependent improvement of collagen metabolism in ligaments by the oral administration of gelatine 60 min before jumping exercises [7
]. This specific time window was based on the fact that peak availability of free and peptide forms of serum hydroxyproline is respectively 1 and 2 h following the oral ingestion of gelatin [38
]. Based on the new insight that the stimulation of exercise-induced anabolic processes might be influenced positively by the oral administration of sCPs immediately before an exercise program [7
], we recommend that future study designs should take absorption kinetics of the available forms of gelatin or sCPs into consideration.
A strength of the present study is that there were only 2 dropouts after T2, 1 in each group, which is a negligible number. Given that our LMM approach operates on a missing at-random-assumption, we deemed the reasons for dropout not violating the assumptions of the model. Furthermore, attrition is low and no individuals were excluded from our LMM so the possibility of selection bias is considered low in the present study. Nevertheless, as the clinical improvements in VISA-A scores after 3 months are in the same range as previously published studies on eccentric calf-strengthening programs [39
], no firm conclusions can be drawn regarding the added benefits of sCP supplementation in chronic Achilles tendinopathy. So, although the present randomized controlled and double-blinded crossover study found a significant interaction of sCP supplementation on change in VISA-A score during a 6-month calf-strengthening plus structured return-to-running program, our findings require duplication in a larger and adequately powered clinical trial.
From a clinical perspective, it is important to note that the majority of our participants had a relatively long history of mid-portion Achilles tendinopathy not responding to rest or other physiotherapy regimes. The latter is also reflected by the fact that placebo group BA only showed a clinically relevant improvement in VISA-A score after they were switched over to sCPs supplementation. This cross-over effect indicates that sCPs supplementation could be a useful adjunct therapy in Achilles tendinopathy patients whom are not responding to a well-standardized eccentric calf strengthening exercise program.
To improve our understanding of the in vivo working mechanism of specific collagen peptides, we also investigated the microvascular changes of the Achilles tendon using a novel medical imaging technique based on contrast-enhanced ultrasonography. The present study is the first of its kind showing that objectively quantified Achilles tendon microvascularity is inversely and moderately associated with Achilles tendon symptoms throughout the course of a therapeutic intervention. The latter indicates that CEUS of tendons may be a useful and more objective medical imaging technique to monitor the clinical response to a therapeutic intervention. Although a previous publication showed that CEUS has a better sensitivity in detecting microvascular abnormalities as compared to power Doppler ultrasound (PDU) [18
], the observed change in CEUS-based microvascularity during the course of the intervention was not associated with the intake of specific collagen peptides. Our rather large inter-subject variability in Achilles tendon microvascularity at baseline may have precluded detecting any statistically significant differences between the 2 groups. Nevertheless, our results are well in line with several other PDU-based imaging studies that reported rather equivocal responses in microvascularity following conservative treatment of Achilles tendinopathy [40
]. Despite the longitudinally observed association between CEUS-based microvascularity and Achilles tendon symptoms, it is unlikely that CEUS will be useful in determining the potential in vivo working mechanism of specific collagen peptides as an adjunct treatment to a well-structured eccentric calf strengthening exercise program.
Eccentric tendon loading has shown to be a safe and effective method to reduce pain and to improve tendon structure [45
]. Although PDU-based studies have been equivocal [40
], eccentric tendon loading has been shown to reduce the number of neovessels in the tendinopathic area of the tendon matrix. The latter is considered to be an important etiological mechanism for the beneficial outcome of a calf strengthening program as also applied in the current study [46
]. Cell-line studies have shown that tenocytes produce the antiangiogenic factor endostatin, a proteolytic fragment of Collagen XVIII, in response to physiological mechanical load, thus limiting neo-angiogenesis [47
]. Although endostatin response to eccentric loading has not been investigated in diseased tendons, several mechanistic studies [47
] indicate that only the right amount of tissue loading increases endostatin expression and, as such, may reduce microvascularity as observed in the present study. As the collagen matrix of tendon is a highly mechanosensitive tissue, cytokine homeostasis and cell survival underlie an intimate balance between adequate biomechanical stimuli and disturbance through load deprivation and overload. This delicate balance between tendon blood flow and loading pattern was recently highlighted in a study by showing that the risk of developing Achilles tendinopathy increased if blood flow increase in the tendon after running was reduced [36
]. Based on these new insights, future studies on novel therapeutic strategies for tendinopathy should preferably assess tendon blood flow response both at rest as well as following a standardized tendon-loading protocol.
Although from a pathophysiological perspective return-to-running status and tendon microvascularity are likely to interact, the limited statistical power of the present pilot study does not allow us to exclude such a significant interaction using linear mixed modelling. The decision to return to running was a function of pain reduction during single leg hopping as well as the successful and pain-free achievement of pre-defined milestones during the course of our rehabilitation program. The latter can be justified by the well-established fact that progressive (and sports-specific) tendon loading is required for successful tendon healing and return to sport [38
]. Furthermore, the milestones described in Appendix A Table A2
entailed significantly lower Achilles tendon loading than present during pre-injury sports participation. Although the VISA-A questionnaires are designed to quantify both activities-of-daily-living (ADL) and running-associated Achilles tendon pain, we cannot exclude that running with incompletely healed tendon pathology may have introduced a certain level of bias towards the overall VISA-A score. As such, for future RCTs we would recommend assessing both ADL-dependent and running sport-dependent Achilles tendon pain on a visual analog scale as secondary outcome measures.
Previous in vitro work on fibroblasts indicates that biosynthesis of ligament and tendon matrix molecules, as well as elastin content can be stimulated by exposure to sCPs [50
]. A recently published shear wave elastography study indicates that, compared to controls, participants with Achilles tendinopathy display lower Achilles tendon elastic modulus [51
]. Accordingly, we recommend that future randomized clinical trials in Achilles tendinopathy patients should include shear wave elastography to monitor changes in tendon elasticity following the intake of sCPs.